1. Specific Aims: Aim 1: Determine the connectivity of ALDH1A1-positive nDA neurons in mouse brain. Identifying how the ALDH1A1-positive nDA neurons are connected to the rest of the brain is instrumental to understanding how these neurons regulate specific behaviors. Genetically encoded neuron and synapse tracers were used to map the projections and monosynaptic inputs of ALDH1A1-positive nDA neurons. Aim 2: Identify the behavioral functions associated with ALDH1A1-positive nDA neurons. Determining the specific behavioral phenotypes regulated by ALDH1A1-positive nDA neurons is essential to understanding the circuit mechanisms of the disease. The activity of ALDH1A1-positive nDA neurons will be manipulated by cell-type specific genetic lesion, optogenetics and chemogenetic approaches. Open-field, accelerating Rotarod, elevated plus maze, and other behavioral tests will be employed to examine both motor and emotional behaviors. Aim 3: Examine the impact of PD-related genetic mutations on the function of ALDH1A1-postive nDA neurons. Adeno-associated viral vectors are used to introduce PD-related genetic mutations in adult mice to avoid potential compensatory responses associated with germline mutations. Live imaging of ALDH1A1-positive nDA neurons in awake, behaving animals will help us monitor potential effects on neuron firing and dopamine release. 2. Scientific Premise and Significance The nDA neurons located in the ventrolateral tier of substantia nigra pars compacta (SNpc), which send their projections mainly into the dorsal striatum, preferentially degenerate in the earlier stages of PD. Recently, our lab demonstrated that this subpopulation of DA neurons can be molecularly defined by selective expression of the ALDH1A1 gene in both human and mouse brains. We further revealed that these ALDH1A1-positive nDA neurons have distinct projection pattern and dopamine release dynamics in the dorsal striatum. In this project, we are working to elucidate how the ALDH1A1-positive nDA neurons are connected to the rest of the brain, how they regulate specific behavioral phenotypes, and how their activity and survival are affected by PD-related mutations. We anticipate the knowledge gained from this study may provide new mechanistic insight into better diagnostic and therapeutic strategies for the treatment of PD. 3. Innovation We are the first to map the circuit connectivity of the ALDH1A1-positive subpopulation of nDA neurons that preferentially degenerate in PD. We found that although ALDH1A1-postive nDA neurons receive inputs from the whole dorsal striatum and many other brain regions, their outputs predominantly converge in the dorsolateral striatum, an area primarily innervated by sensorimotor cortices, suggesting a critical involvement of ALDH1A1-positive nDA neurons in motor control. In support of this notion, our behavioral experiments revealed a critical contribution of ALDH1A1-positive nDA neurons to enforce motor skill acquisition and retention. Aberrant motor skill learning is implicated in PD-related motor impairments24. Our studies uncover the potential underlying circuit mechanisms. 4. Research Plan In Aim 1, to genetically manipulate ALDH1A1-positive nDA neurons, we generated a new line of Aldh1a1-P2A-CreER (Aldh1a1+/CreER) knock-in (KI) mice, in which Cre recombinase is expressed under the control of the endogenous Aldh1a1 promoter. By crossing with a Cre-dependent tdTomato reporter line (Ai9), we confirmed the uneven projection pattern of ALDH1A1-positive axon fibers in the dorsal striatum as revealed previously by anti-ALDH1A1 antibody staining. To identify the neurons that directly innervate ALDH1A1-positive nDA neurons, we mapped the monosynaptic inputs using rabies labeling. We found that ALDH1A1-positive nDA neurons receive the majority of inputs from striatal neurons. In comparison to previously reported data that mapped the monosynaptic inputs to both ALDH1A1-positive and negative nDA neurons, the ALDH1A1-positive DA neurons receive less innervation from the cortex, but more projections from zona incerta (ZI) and ventral striatum. ZI is one of the top clinical targets for deep brain stimulation (DBS), a surgical procedure to relieve PD symptoms. Together, we produced the first comprehensive input/output circuit map of ALDH1A1-positive DA neurons in the mouse brain. In Aim 2, to elucidate the function of ALDH1A1-positive nDA neurons, we genetically ablated these neurons in Aldh1a1CreER knock-in mice by stereotaxic injection of AAVs expressing Cre-dependent apoptotic gene caspase 3 (taCasp3). The ablation of ALDH1A1-postive nDA neurons did not affect spontaneous movements in the Open-field tests, but compromised motor learning in the accelerating Rotarod test, suggesting a critical involvement of ALDH1A1-positive nDA neurons in the acquisition and maintenance of skilled movements. To determine if the learning deficits resulted from loss of dopamine transmission, we will treat the mice with L-DOPA and repeat the motor learning task. To further determine the function of ALDH1A1-positive nDA neurons in movement acceleration and deceleration, we infected the neurons with AAVs carrying Cre-dependent optogenetic activators and inhibitors. Our preliminary results found activation of ALDH1A1-positive nDA neurons modestly increased general locomotion. We will next examine the effects of acute neuron inhibition on locomotion. In Aim 3, to evaluate the impact of PD-related genetic mutations on the function of ALDH1A1-postive nDA neurons, we injected Aldh1a1CreER knock-in mice with AAVs that contain Cre-dependent alpha-synuclein A53T mutation. Although the ectopic expression of alpha-synuclein did not cause any overt neuropathological abnormalities or locomotion impairments, our preliminary data revealed similar deficits in motor learning. We will attempt to rescue this phenotype with L-DOPA, and also perform live imaging of ALDH1A1-positive nDA neurons in awake, behaving animals to monitor neuron firing and dopamine release. Additionally, we will inject Aldh1a1CreER knock-in mice with AAVs that express Cre-dependent CRISPR/Cas9 vectors to knockout DJ-1, Parkin, and PINK1, and test the mice as outlined above.